The goal of this project is the description of structural features that are responsible for recognition and reactivity in proteins, using high-resolution x-ray structure analysis as a primary method. The structure determinations focus on a series of flavin- and metal-containing enzymes and employ genetic engineering, spectroscopic, and biochemical techniques, in combination with x-ray crystallography, to address such questions as the control of redox equilibria by proteins. In continuing studies of flavodoxins, which are electron-transferring proteins that utilize the FMN cofactor, site-directed mutants and flavodoxins with modified flavins will be studied to evaluate the role of electrostatic interactions in producing the very low potentials which are typical of these proteins. The structure of phthalate dioxygenase reductase (PDR), a protein from Pseudomonas which mediates electron transfer from FMN to a [2Fe-2S] center, will be refined and determined in its reduced states and in complexes with pyridine nucleotides. The spacing and orientation of the FMN and [2Fe-2S] centers within the PDR protein make it an novel subject for studies of electron transfer. Mutants and metal-ligated species of Fe- and Mn- containing superoxide dismutases will be examined to understand the structural chemistry of the metals in these oxygen scavengers and to assess proposed mechanisms for the coupling of electron with proton uptake. The a priori determination of the three-dimensional structures of lactate oxidase from M. smegmatis and methionine synthase from E. coli will be undertaken to answer questions about protein-substrate interactions, reaction mechanisms, and regulation in these two complex enzymes. Lactate oxidase in an octamer of Mr 350,000 and methionine synthase is one of the largest enzyme polypeptides, with Mr 130,000. Mammalian methionine synthase, an analog of the E. coli enzyme, is one of the enzymes associated with the manifestations of B12 deficiency, and a potential chemotherapeutic target. Attempts will be made to crystallize mammalian cytochrome P-450s and metal-regulated activators of transcription.